1. Field of the Invention
This invention relates generally to compositions and methods for labeling molecules, and more specifically to small, synthetic molecules that react with target sequences.
2. Background Information
Many techniques in the biological sciences require attachment of labels to molecules, such as polypeptides. For example, the location of a polypeptide within a cell can be determined by attaching a fluorescent label to the polypeptide.
Traditionally, labeling has been accomplished by chemical modification of purified polypeptides. For example, the normal procedures for fluorescent labeling require that the polypeptide be covalently reacted in vitro with a fluorescent dye, then repurified to remove excess dye and/or any damaged polypeptide. Using this approach, problems of labeling stoichiometry and disruption of biological activity are frequently encountered. Furthermore, to study a chemically modified polypeptide within a cell, microinjection can be required. These processes can be tedious and typically cannot be performed on a large population of cells.
Thiol- and amine-reactive chemical labels exist and can be used to label polypeptides within a living cell. However, these chemical labels are promiscuous. Such labels cannot react with a particular cysteine or lysine of a particular polypeptide within a living cell that has numerous other reactive thiol and amine groups.
Another method of intracellular labeling of polypeptides in living cells has involved genetically engineering fusion polypeptides that include green fluorescent protein (GFP) and a polypeptide of interest. However, GFP is limited in versatility because it cannot reversibly label the polypeptide. In addition, GFP is a full size protein of 238 amino acids. GFP's large size frequently perturbs the protein interest upon binding. In addition, the spectroscopic read-out for GFP is at an emission maxima of up to 529 nm. Although red emitting fluorescent proteins are known to the art, their development has been slow and their utility has been greatly restricted.
Recently, another method of intracellular labeling of polypeptides in living cells wherein a fluorescent biarsenical compound binds to a tetracysteine motif having the sequence Cys-Cys-Xaa-Xaa-Cys-Cys (SEQ. ID NO: 1) (wherein Xaa is any amino acid other than cysteine). C. Griffin, et al., science 1998, 281, 269-272; U.S. Pat. Nos. 6,451,569 B1, 6,008,378, 6,054,271, and 5,932,474, all of which are herein incorporated by reference. The Cys-Cys-Xaa-Xaa-Cys-Cys (SEQ ID NO: 1) motif occurs infrequently in nature such that recombinant addition of this motif to a target protein provides a selective method of functionally tagging a defined protein. However, additional motifs which occur infrequently in nature and are capable of binding biarsenical molecules would be useful.